Fig 2: (A,B) MTT-based cell proliferation assay and ROS detection assay (0–24–48–72–96h) on untreated C2C12 cells (CTRL), C2C12 cells treated with vehicle of FLAVOmega ß (CTRL DMSO), C2C12 treated with formulation FLAVO a, FLAVO ß, FLAVO ?, FLAVOmega a, FLAVOmega ß and FLAVOmega ? (n = 3 for each experimental group). Relative luminescence units were measured by GloMax Discover System (Promega Corporation, USA) at 570 nm. (B) Two-way ANOVA followed by Tukey’s multiple comparison test: C2C12 cells treated with FLAVOmega ß compared to CTRL DMSO (** p = 0.0040) at 72 h after treatment; C2C12 treated with FLAVOmega ß compared to CTRL (** p = 0.0048) and CTRL DMSO (** p = 0.0046) at 96 h. All values are expressed as the mean ± SD. (C) Representative image of FLAVOmega ß formulation. (D) Absorption spectrum of FLAVOmega ß formulation (top left); time-resolved PL of FLAVOmega ß formulation (top right); dynamic light scattering (DLS) analysis of FLAVOmega ß formulation (bottom).

Fig 3: Comparison of sensitivity of [Ru(bpy)2(dabpy)]2+ with direct and indirect conventional NO assays in cell-free media. Nitric oxide concentration dependent changes in the ?fluorescence calculated as a percentage from the baseline reading in the absence of NO (0 µM NOC13) for (a) [Ru(bpy)2(dabpy)]2+ at ?ex = 450 nm and ?em = 615 nm and for (b) DAF-FM-diacetate at ?ex = 490 nm and ?em = 515 nm. Comparable changes in the ?absorbance at ?ab = 560 nm calculated as a percentage from the baseline reading in the absence of NO, derived from the Griess assay for (c) nitrites only and (d) total nitrites + nitrates. Each bar represents the mean percentage ± s.d of four independent experiments and the symbols represent the mean percentage of 2–3 replicates in each experiment in cell free PBS, using the same spectrophotometer (Glomax Discover System, Promega). The NOC13 concentrations were adjusted to account for the relevant time points in each assay and to compare the response at each concentration of NO. Only p-values less than 0.05 are reported, based on one-way ANOVA followed by Dunnett’s multiple comparisons test to 0 µM NO.

Fig 4: Relative luminescence units (RLUs) produced by HT-29 cells during (A) and at the end (B) of Promega’s RealTime-Glo™ MT Cell Viability assay. HT-29 cells (1,500 cells per well) were incubated in DMEM High glucose medium supplemented with 10% fetal bovine serum (FBS) and antibiotic–antimycotic at 37°C in a humidified 5% CO2 atmosphere. Cells were supplemented either with H2Opure (?), bortezomib (?), or varying concentrations of 5-KF (?1 mM, ? 5 mM, ? 10 mM, ¦ 20 mM, ? 30 mM). Reagents of Promega’s RealTime-Glo™ MT cell viability assay were added according to the manufacturer’s instructions. At 10 individual time points, the relative luminescence units (RLUs) were assessed using a Promega Glomax® Discover System. Tukey’s multiple comparison test was performed using GraphPad 9.3.1 (GraphPad Software, San Diego, US) to determine statistical significance; ****p < 0.0001. The experiment was performed using six biological replicates for each test compound.

Fig 5: Endocytic entry of R-909 into J-HER2 and SK-OV-3 cells.(A-C) J-HER2 (A), SK-OV-3 (B), or J-nectin1 (C) cells were infected with the indicated viruses, in the presence of increasing nM concentrations of Bafilomycin A (BFLA). The extent of infection of GFP-positive viruses was determined by GloMax Discover System (Promega), and that of R-809 was determined by FACS. Each point represents the average of triplicate samples ± S.D.